Isolation Identification and Antimicrobial Susceptibility Pattern of Salmonella and S Aurous from Selected Dairy Farms in Beedle and Nekemte Districts Western Ethiopia

Introduction

Food-borne disease is common in developing countries, particularly in Africa, due to inadequate food safety regulations, a weak regulatory framework, a lack of financial resources to invest in safer equipment, and a lack of knowledge of food-handler [1]. Changes in eating habits, mass catering, hazardous food storage conditions, and poor hygiene practices all contribute to the spread of food-borne illnesses. Contaminated feces, eggs, meat, milk, and milk products all contribute to the spread of zoonotic pathogens. Contaminated feces, eggs, meat, milk, and milk products all increase the risk of zoonotic pathogen transmission [2]. Foodborne diseases are a public health issue in both developed and developing countries. Over 250 different foodborne diseases have been identified. The majority of these diseases are caused by infections produced by various bacteria, viruses, and parasites. Poisonings are another type of disease caused by hazardous poisons or substances, such as poisonous mushrooms [3]. In countries where foodborne illness was investigated and documented, pathogens such as S. aurous, Campylobacter, E. coli, and Salmonella species were identified as prominent causes. These organisms were previously known to induce acute gastroenteritis and may produce a more serious septicemia disease, usually in the very young, the elderly or immune-weakened individuals [1].

Foodborne bacterial diseases pose a significant threat to human and animal health. Salmonella is one of zoonotic food- borne pathogen that may be found in food. Salmonellosis is the most prevalent foodborne bacterial disease in the world, causing severe bacterial enteric disease in both humans and animals [4]. Increasing proportion Salmonella isolates currently demonstrate resistance to various antimicrobial drugs in both developing and developed countries. Salmonellosis in animals is classified into several subcategories. Salmonella abort us ova’s in sheep, Choleraesuis in pigs, Gallinarum in poultry, abort us equip in horses, and Dublin in cattle are the primary Salmonella samovars that cause disease. These samovars are primarily responsible for abortions or acute gastroenteritis in their hosts. Salmonella samovars that cause disease in several animal and human hosts include S. Typhimurium, S. Enteritis, S. Hadar, and S. Infantis [5]. The use of antimicrobial agents in food animals leads to the emergence of antibiotic-resistant salmonellae, which are zoonotic bacteria and are transferred to humans, typically through the food supply [6].

Escherichia coli is a common food contaminant and a good sign of fecal contamination. The presence of E. coli in milk products suggests the existence of enter pathogenic microorganisms that pose a risk to public health. [7] E. coli is one of several pathogenic bacteria that can contaminate milk and some dairy products, and it is regarded as a reliable sign of contamination by manure, soil, and contaminated water [8]. E. coli is a natural inhabitant of animal and human intestines, but its recovery from food may be a public health issue because of the existence of enters pathogenic and/or toxigenic strains that cause severe gastrointestinal disturbances. Other toxigenic bacteria, such as E. coli O157: H7, induce life-threatening syndromes [9]. Staphylococcus aureus is famous for developing antibiotic resistance. The development of multidrug resistance in S. Aureus is a global issue. Because of its ability to form an exopolysaccharide capsule, S. Aureus develops drug resistance more rapidly, and its location in the micro abscess limits drug access to infected cells [10]. Milk and dairy products have been linked to diseases associated with milk collection and normal processing conditions, which may allow bacteria in dairy cows and the dairy environment to be introduced directly into the milk. The highly nutritious milk medium, once introduced, promotes fast microbial growth. As a result, the risk of food-borne disease and intoxication from milk and dairy products is a problem [11].

Antimicrobial resistance is becoming an increasingly serious danger to global public health. In developing countries, the situation is worse because, in addition to the increased use of antibiotics and their easy availability without a prescription, inadequate sanitation surrounding premises contributes to the development of resistant strains. It is now well established that clinically significant bacteria are defined not only by a single drug resistance but also by multiple antibiotic resistance [10]. The increased use of antibiotics in the poultry, fishery, and livestock production industries to treat and prevent infections, or as growth promoters has greatly contributed to the growth in antibiotic resistance in potential food-borne pathogens (Salmonella, E. coli, S. Aureus, Shigella, Campylobacter, and etc.) in past years. The widespread use of antibiotics in agriculture has contributed significantly to the emergence and spread of antibiotic-resistant food-borne pathogens in humans as a result of poultry and dairy product consumption [10].

Foodborne infections have been identified as a significant public health and economic hazard in both developed and developing countries. As a result, microbial food safety has developed as a major global concern for consumers, industry, researchers, and regulatory authorities. Microbial contamination is one of the primary causes of food spoilage throughout the world [12]. The World Health Organization (WHO) estimates that up to 30% of the population in developed countries suffers from food-borne diseases each year, while up to 2 million deaths are estimated in underdeveloped countries [13]. Food-borne diseases are frequent in underdeveloped nations like Ethiopia due to poor food handling and sanitation practices; lack of food safety regulations; weak regulatory systems; a lack of financial resources to invest in safer equipment; and a lack of education for food handlers [14]. In recent years, a number of studies in Ethiopia have observed the occurrence of Escherichia coli in foods of animal origin, primarily meat and milk [15, 16, 17, 18]. Because of the large animal extensive farming methods in those locations, the majority of the investigations were done in central Ethiopia. Estimation and quantification of Escherichia coli occurrence at the national level might assist responsible entities in the prevention and control of its occurrence in foods before they reach end consumers, hence decreasing its effect [7].

In Ethiopia, the first published antimicrobial preliminary study on AMR for several microbial agents was published in 1970. Since then, AMR reports from various antimicrobial surveillance and research have revealed the fast development and dissemination of resistant strains. According to Sibhat, et al. [19] (20.7%) of the 87 isolates of Salmonella serovars Newport (n = 14), Anatomy (n = 3) and East Bourne (n = 1) were resistant to two or more antimicrobials. S. Newport was multidrug resistant (15.6%) and resistant to streptomycin, sulphisoxazole, and tetracycline among the antimicrobial resistant Salmonella serovars [20]. Staphylococcus aureus isolates from bovine mastitis milk exhibit significant levels of resistance to ampicillin, penicillin, polymixin B, and streptomycin [21, 22, 23, 24].

Due to the substantial proportion of Ethiopians who live in close proximity to their animals, there is a risk of the transfer of resistant Staphylococcus aureus from animal to human via milk intake. To facilitate more suitable treatment decisions, reduce morbidity and mortality related to resistant infections, and maintain antibiotic efficacy, the knowledge of AMR in a country must be summarized and synthesized. Updating national treatment recommendations requires evidence that has been appropriately summarized and synthesized [25].

Another major concern for human health today is antimicrobial resistance caused by antibiotic usage in livestock production, as well as human disease situations in developing countries. Penicillin, streptomycin, gentamycin, and oxy tetracycline are the most common antibiotics used in Ethiopia to treat animal and human infections [25, 26]. Despite the need for a better understanding of antibiotic use in Ethiopia, this resistance variation may be due to the indiscriminate use of antimicrobials in animal production without prescription in the animal and human health sectors, which may favor selection pressure that increased the advantage of bacteria maintaining resistance genes [26]. Ethiopia is one of the countries that continue to use antimicrobial drugs in dairy production, mostly for therapeutic purposes [25]. Unnecessary use of antimicrobial drugs in dairy production has been speculated to induce selection pressure for antimicrobial resistance; this method can be transmitted from animals to human primarily through the food chain. Antibiotic selection pressure allows the microbe to survive as a resistant strain. Not only will resistant bacteria grow and produce resistant offspring, but they may also horizontally transfer the resistance gene to other microbes in other hosts and geographic regions [27].

Antibiotic resistant microorganisms spread from animal to human via food. Because antibiotic-resistant bacteria can be transferred to humans through food supply owing to unsanitary and conventional milk production and processing procedures, the problem of milk safety remains a difficulty. If these safety concerns are not addressed, the high nutritional makeup and neutral pH of milk may transport numerous antibiotic resistant foodborne bacteria [26]. As a result, antibiotic resistance linked with foodborne pathogens should be closely examined in this sector. However, there is little information on the isolation and identification of foodborne salmonella species, E. coli, and S. aurous, as well as their antibiotic susceptibility pattern in western Ethiopian dairy cows. As a result, the study focused on the isolation, identification, and antibiotic susceptibility profile of selected foodborne bacterial pathogens linked to dairy cow production in western Ethiopia.

Biochemical Characterization of Salmonella Isolates

Biochemical assays are used to identify Salmonella isolates. L-lysine decarboxylation, Indole, Methyl red, Voges proskaeur. The isolates were then subjected to the following procedures to determine their biochemical activity. On TSI, urea hydrolysis and H2S generation are seen [31].

Isolation and Identification of Staphylococcus Species

The process flow chart shows how Staphylococcus aureus was isolated and identified. Gram’s staining, catalase test, sugar fermentation, and coagulase tests are used to validate the presumptive identification of Staphylococci organisms [30].

Coagulase Test

In sterile tubes, the tube coagulase test is conducted. The chosen Staphylococcus was sub cultured into brain heart infusion broth and incubated for 24 hours at 37 degrees Celsius. Then, 0.5 ml of broth culture and 0.5 ml of sterile rabbit plasma were combined in a thin sterile tube, along with a reference tube containing 0.5 ml of sterile Brain Heart Infusion broth and 0.5 ml of rabbit plasma, and incubated at 37 o C for 4 and 24 hours, with clot formation noted. When compared to the control, any coagulation of plasma is considered positive at any of the values [30].

Antimicrobial Susceptibility Test

The antimicrobial susceptibility test was performed according to the Clinical and Laboratory Standards Institute’s agar disc diffusion technique. In brief, the bacteria are suspended in 0.85 percent sterile normal saline solution in a 0.5 Mac-Farland standardized suspension. A sterile cotton swab was dipped in the standardized bacteria suspension and then streaked uniformly across the Mueller-Hinton agar surface. The paper discs are then put on the agar surface, each impregnated with a specific concentration of antibiotics. The susceptibilities of the isolates were tested for the following antibiotic discs: Ceftriaxone (30μg), Ciprofloxacin (5μg), Gentamicin (10μg), Nalidixic acid (30μg), Streptomycin (10μg), Cefoxitin (30μg), Penicillin G (10μg), Azithromycin (30μg), Nitrofurantoin (300μg), Erythromycin (15μg) and Tetracycline (30μg) were placed at least 15 mm apart and from the edge of the plates to prevent overlapping of the inhibition zones and incubated in an inverted position at 37°C for 24 hours. After incubation for 24 hours, clear zones of inhibition are produced by the bacterial growth and diffusion of the antibiotics and these were measured in millimeter using a caliper and interpreted as susceptible, intermediate and resistant.33 The antimicrobial susceptibility of Enterobacteriaceae and S. Aureus to different drugs is presented in table 1 and 2 below.

Key: S, Susceptible; I, Intermediate; R, Resistance Table 1: Antimicrobial susceptibility test interpretive criteria for Enterobacteriaceae [33].

Key: S, Susceptible; I, Intermediate; R, Resistance Table 2: Antimicrobial susceptibility test interpretive criteria for S. Aureus [33].

Data Analysis

Data were entered to a Microsoft Excel spreadsheet and analyzed by using SPSS software package (SPSS 20.0 for window 7, SPSS Inc, Chicago, Illinois). Descriptive analysis is used to describe the result of proportion analysis. Proportion is estimated as the number of samples detected positive to Salmonella, E. coli and S. Aureus isolated from the total sample analyzed. Chi-square test was done to study association between Salmonella, E. coli and S. aurous isolates and variables considered (sampling area, sample source, and sample type). The significance level was set at 0.05.

Discussion

Infection with Salmonella, E. coli, and S. Aureus in dairy cattle is still a big issue across the world. Significant economic losses were incurred as a result of infected animals’ mortality and poor growth, as well as the danger of transmission to people via the food chain or direct animal contact. As a result, detecting animals contacting humans and equipment is critical for controlling Salmonella, E. coli, and S. Aureus on- farm and their spread to the general public [2, 34, 35].

The frequency of Salmonella in apparently healthy lactating dairy cows (udder milk & fecal samples) in this research (7.9%) is lower than in a similar report from Gondar town (12.5%) [36]. Even if the finding is lower, lactating cows are potential sources of Salmonella infection for dairy farm workers and the general public. The current investigation found a fecal Salmonella prevalence of 3.97% among lactating dairy cattle, which is lower than the fecal Salmonella isolation rate of 7.7% in lactation cows and in contact people in Addis Ababa dairy farms [37, 38].

The current result also agrees with a report of a 1.56% prevalence of Salmonella fecal shedding in dairy cattle in Egypt by Mohamed, et al. [39] and 0.7 percent in dairy cows in the United States (USA) by Callaway et al. [40]. The current investigation also demonstrated that the variation in the volume and relative occurrence of Salmonella isolates between the current and previous studies in different locations of Ethiopia might be related to differences in risk variables that lead to Salmonella occurrence. These are host- related risk variables such as age, breed, animal physiological condition, feeding techniques, and immunization status [35, 41]. Environmental risk factors include hygiene and management practices; stocking density; feed type and volume; readily available water sources; use of contaminated utensils; and housing [42].

Although a relatively low proportion of Salmonella (2.35%) was isolated and identified in the current study compared to previous studies, this could pose significant health risks to humans and animal species by provoking salmonellosis in high-risk groups such as newborns, infants, the elderly, and immune compromised individuals who are susceptible to Salmonella infections at a lower infective dose than healthy adults. As a result, it is a source of Salmonella infection through the eating of infected dairy products, which is particularly significant in Ethiopia, where dairy products are regularly ingested without appropriate boiling.

In Ethiopia, E. coli is regarded as a major threat to dairy development and public health. Escherichia coli is regarded as not only a sign of fecal contamination but also of inadequate hygiene and sanitary practices during milking and subsequent handling. This study also suggests that E. coli is the most significant dairy development obstacle in the study area. The frequency of E. coli in lactating cows, milkers, and dairy farm environments was reported to be 11.75% in the Bedele and Nekemte districts. This research is lower than the findings of Mohanty, et al. which found (21%) from India [43].

The current finding was in agreement with the findings of Lye, et al. [44] and Addo, et al. [45], who reported 8.75% and 11.2 %, respectively, from Malaysia and Ghana. The present finding, however, is higher than the one published by Bedasa, et al. [46], who reported a prevalence of 3.5% in food of animal origin in Bishoftu, Central Ethiopia. This difference in findings between the current study and previous investigations might be related to differences in environmental conditions, management, and hygiene standards. This study was also undertaken to determine the frequency of Staphylococcus aureus in dairy farms, as well as their antibiotic susceptibility. The capacity of Staphylococcus aureus to appear in a variety of environments may disclose their resilience properties. Bucket swab (5%), udder milk (2.97%), fecal (0.99%), floor swab (5%), and cow nasal swabs (1.98%) were among the samples collected in diary settings. The results of the current investigation were consistent with those reported by Bitew, et al. [47] in Bahir Dra, 3.9 percent in Adama by Abera, et al. [48], and 6.25 percent in Egypt by Thaker, et al. [49].

The current finding was lower than the report of Abebe, et al. (15.5%) [50] and Reta, et al. (24.2%) [51] On S. Aureus in raw milk samples in Ethiopia. El-Gedawy, et al. found 51% and 17% in Egypt, and 52% and 18.2% in Turkey, according to Ekici, et al. [52]. This variation in S. Aureus prevalence between reports could be attributed to differences in farm management practices, study methods, and agro-climatic conditions, as well as the fact that milk always contains microorganisms derived from the milk ducts in the udder, even when drawn under aseptic conditions. Furthermore, pollutants from milking tools, human handlers, dirty ambient conditions, and improper udder preparation may cause bacterial contamination of raw milk.

Antimicrobial use in animal production systems has long been suspected to a cause of the emergence and dissemination of antimicrobial resistant pathogens. Improper use of antimicrobials in both human and veterinary medicine has contributed to development and dissemination of antimicrobial resistant pathogens [35, 37]. In current study, resistance to three or more of antimicrobials was observed only on one Salmonella isolate resistant to gentamycin, ciprofloxacin, cefoxitin and tetracycline. This is lower than studies conducted in Ethiopia by Addis, et al. (10.7%) [37] and Tadesse and Dabassa (80% to Nalidixic acid, 35% each to Tetracycline and Kanamycin and 30% to Amikacin Gentamycin, 25% each to Chloramphenicol and Streptomycin and 5% to Ciprofloxacin) [53] and Fadlalla, et al. (46.8% to 10 antimicrobial drugs) [54] in Sudan. A study in Alexandria Egypt by Mohamed, et al. [39] reported that 85.7% of Salmonella species isolated from dairy cattle were sensitive to tetracycline. This result agrees with the current study in which 77.78 % of the isolates were sensitive to tetracycline. This study also reported that 100% of Salmonella species isolated from dairy cattle were sensitive to nalidixic acid. This finding is also disagreed with a report in Sudan by Fadlalla, et al. [54] in which Salmonella isolates from human and cattle were 100% susceptible to ciprofloxacin whereas our current study found 55.5% of the tested Salmonella was susceptible to ciprofloxacin.

In current study all E. coli isolates were found to be 100% susceptible to nitrofurantoin, ceftriaxone, nalidixic acid and ciprofloxacin followed by 90.47%, 76.19%, 71.42% and 61.9% of the isolates were susceptible to cefoxitin, gentamicin, streptomycin and tetracycline, respectively (table 5). Current study is relatively in line with findings reported by Reuben and Owen [55] who reported all E. coli isolates were 89.5% of the isolates were susceptible to gentamicin from Nigeria. Bagre, et al. [56] found all E. coli isolates were 100% susceptible to gentamicin, from Burkina Faso. Salehi and Bonab [57] also reported all E. coli isolates were 100% susceptible to gentamicin from Iran. Similar studies conducted in Ethiopia by Tesfaye, et al. [58] and in Nigeria by Wariso, et al. [59] found similar susceptibility rates with current study. High sensitivity to ciprofloxacin, gentamicin and nitrofurantoin, have been recorded from previous studies conducted in India [60]. In this study, nitrofurantoin, ceftriaxone, nalidixic acid and ciprofloxacin were found to be the most effective antimicrobials against E. coli isolates. The overall resistance of E. coli isolates in this study was low this indicates antimicrobial drug usage of the study area may be lower than the other areas, due to agro- climatic condition of the areas.

Table 6 summarizes the antimicrobial resistance profile of S. Aureus isolates from cow milk, milk-related equipment, and farm floor swab samples. As a result, all S. Aureus isolates in this investigation were completely resistant to cefoxitin, nitrofurantoin, and gentamicin. According to some investigators, the increasing incidence of penicillin G resistant isolates might be linked to extended, inappropriate, and indiscriminate use [53]. The resistance pattern of S. Aureus to penicillin G and erythromycin was found to be 55.6% and 11.1%, respectively, in the current study, which is lower than the findings of Sori, et al. (87.2%) [61] In Ethiopia and Landin (80%) [62]. In Sweden, but strongly agrees with Gooraninejad, et al. (57%) in Iran [63].

This study also disagreed with reports from other researchers indicated S. Aureus isolates were resistance to ciprofloxacin (90.9%), ceftriaxone (63.6%), and Penicillin-G (81.8%). Similar result was most frequently observed for penicillin (100%) followed by erythromycin (Wang, et al. (95.7%) [64]. In China, penicillin G by Beyene (100%) [65]. In Ethiopia, whereas the percent were found higher when compared to report by Hamid, et al. [66] of 94.4 and 50% resistance for penicillin and ceftriaxone, respectively of S. Aureus isolates with particular emphasis on penicillin G. The possible explanations for the high record of most drug Susceptible S. Aureus in dairy farms may be due to the limited use of antibiotics in dairy farms of the study area [67, 68].

Conclusion and Recommendations

Food-borne bacterial infections, such as Salmonella, E. coli, and S. aurous, are the most common causes of disease in humans and animals when contaminated foods are consumed, particularly dairy products. Infected milk had a higher prevalence of E. coli isolates than salmonella and S. aurous isolates. E. coli isolates were shown to be extremely sensitive to nitrofurantoin, ceftriaxone, nalidixic acid, and ciprofloxacin whereas just a few isolates were resistant to tetracycline and streptomycin based on antimicrobial susceptibility patterns. When compared to previous research in other places, there was a reduced incidence of S. aurous.

S. Aureus were 100% sensitive to cefoxitin, nitrofurantoin, and gentamicin. Occurrence of Salmonella at dairy farms level showed that dairy cattle and their environment are important source of contamination. Nalidixic acid could be considered as first choice drugs as the isolates of Salmonella were susceptible to this drug. Even if, the current finding show that lower prevalence and lower antimicrobial resistance, it is better to give attention for further improvement of hygienic measures as well as antimicrobial drug usage of dairy farms, to safeguard the public from risk of S. aurous, E. coli and Salmonella pathogens which are causing food poisoning and acquiring multi drug resistant isolates in study area. Because many people regularly drink raw milk with no further heat processing, it is a critical public health concern because milk is a vehicle for food-borne diseases.

Based on the abovementioned conclusion, the following recommendations should be considered: Everyone involved in the milk and dairy production chain should be certified in sanitary procedures to ensure raw milk quality.

• To protect customers from zoonotic infections, thorough animal and environmental hygiene should be followed in order to break the bacteria’s continued transmission in farms
• Physicians in the area should consider nitrofurantoin, ceftriaxone, nalidixic acid and ciprofloxacin as first choice of drugs in the treatment of clinical diseases associated with E. coli also cefoxitin, nitrofurantoin, and gentamicin for S. Aureus and nalidixic acid for Salmonella.
• In order to prevent antimicrobial resistance in the study area physicians as well as veterinarians should have to create awareness for dairy farm owners about antimicrobial resistance occurrence and its effect.

Availability of Data and Materials

On reasonable request, the corresponding author made the data sets utilized in the current study accessible.

Ethical Clearance

Animal study ethical clearance was obtained from Addis Ababa University college of veterinary medicine, with reference number VM/ERC/01/06/10/2018. All of the animal owners and attendant were informed about the purpose of the study, as well as the significance and benefit of the research in terms of immediate and future values. While collecting samples, safe handling protocols were followed.

Acknowledgements

Our sincere thanks go to AMR-TR project entitled “Improving Surveillance of Antimicrobial Resistance of Selected Foodborne Zoonotic Bacteria in Central and Western Ethiopia: Risk Factors, Molecular Epidemiology and Ecology, and Interventions Approach” funded by the Addis Ababa University for financial support during the study period. We would also like to acknowledge Bedele and Nekemte districts Animal health bureau. We are also greatly indebted to farm owners who without their willingness we couldn’t have accomplish this work. Finally, we would like to thank Animal Health Institutes and Bedele Regional Veterinary Laboratory for providing the materials utilized for the testing.

Author Contributions

All authors made significant contributions to the conception and design, data acquisition, data analysis, and data interpretation; participated in the drafting of the article or critically revised it for important intellectual content; agreed to publish in the current journal; and gave final approval of the version to be published.

Disclosure

The authors disclose no conflicts of interest in this study.